Karen L. Maxwell

Spatially controlled simultaneous patterning of multiple growth factors in three-dimensional hydrogels

Three-dimensional (3D) protein-patterned scaffolds provide a more biomimetic environment for cell culture than traditional two-dimensional surfaces, but simultaneous 3D protein patterning has proved difficult. We developed a method to spatially control the immobilization of different growth factors in distinct volumes in 3D hydrogels, and to specifically guide differentiation of stem/progenitor cells therein. Stem-cell differentiation factors sonic hedgehog (SHH) and ciliary neurotrophic factor (CNTF) were simultaneously immobilized using orthogonal physical binding pairs, barnase-barstar and streptavidin-biotin, respectively. Barnase and streptavidin were sequentially immobilized using two-photon chemistry for subsequent concurrent complexation with fusion proteins barstar-SHH and biotin-CNTF, resulting in bioactive 3D patterned hydrogels. The technique should be broadly applicable to the patterning of a wide range of proteins.

Structural proteomics of an archaeon.

A set of 424 nonmembrane proteins from Methanobacterium thermoautotrophicum were cloned, expressed and purified for structural studies. Of these, ∼20% were found to be suitable candidates for X-ray crystallographic or NMR spectroscopic analysis without further optimization of conditions, providing an estimate of the number of the most accessible structural targets in the proteome. A retrospective analysis of the experimental behavior of these proteins suggested some simple relations between sequence and solubility, implying that data bases of protein properties will be useful in optimizing high throughput strategies. Of the first 10 structures determined, several provided clues to biochemical functions that were not detectable from sequence analysis, and in many cases these putative functions could be readily confirmed by biochemical methods. This demonstrates that structural proteomics is feasible and can play a central role in functional genomics.

Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system

A widespread system used by bacteria for protection against potentially dangerous foreign DNA molecules consists of the clustered regularly interspaced short palindromic repeats (CRISPR) coupled with cas (CRISPR-associated) genes. Similar to RNA interference in eukaryotes, these CRISPR/Cas systems use small RNAs for sequence-specific detection and neutralization of invading genomes. Here we describe the first examples of genes that mediate the inhibition of a CRISPR/Cas system. Five distinct ‘anti-CRISPR’ genes were found in the genomes of bacteriophages infecting Pseudomonas aeruginosa. Mutation of the anti-CRISPR gene of a phage rendered it unable to infect bacteria with a functional CRISPR/Cas system, and the addition of the same gene to the genome of a CRISPR/Cas-targeted phage allowed it to evade the CRISPR/Cas system. Phage-encoded anti-CRISPR genes may represent a widespread mechanism for phages to overcome the highly prevalent CRISPR/Cas systems. The existence of anti-CRISPR genes presents new avenues for the elucidation of CRISPR/Cas functional mechanisms and provides new insight into the co-evolution of phages and bacteria.

Protein folding : Defining a "standard" set of experimental conditions and a preliminary kinetic data set of two-state proteins

Recent years have seen the publication of both empirical and theoretical relationships predicting the rates with which proteins fold. Our ability to test and refine these relationships has been limited, however, by a variety of difficulties associated with the comparison of folding and unfolding rates, thermodynamics, and structure across diverse sets of proteins. These difficulties include the wide, potentially confounding range of experimental conditions and methods employed to date and the difficulty of obtaining correct and complete sequence and structural details for the characterized constructs. The lack of a single approach to data analysis and error estimation, or even of a common set of units and reporting standards, further hinders comparative studies of folding. In an effort to overcome these problems, we define here a “consensus” set of experimental conditions (25°C at pH 7.0, 50 mM buffer), data analysis methods, and data reporting standards that we hope will provide a benchmark for experimental studies. We take the first step in this initiative by describing the folding kinetics of 30 apparently two‐state proteins or protein domains under the consensus conditions. The goal of our efforts is to set uniform standards for the experimental community and to initiate an accumulating, self‐consistent data set that will aid ongoing efforts to understand the folding process.

Multiple mechanisms for CRISPR–Cas inhibition by anti–CRISPR proteins

The battle for survival between bacteria and the viruses that infect them (phages) has led to the evolution of many bacterial defence systems and phage-encoded antagonists of these systems. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated (cas) genes comprise an adaptive immune system that is one of the most widespread means by which bacteria defend themselves against phages. We identified the first examples of proteins produced by phages that inhibit a CRISPR–Cas system. Here we performed biochemical and in vivo investigations of three of these anti-CRISPR proteins, and show that each inhibits CRISPR–Cas activity through a distinct mechanism. Two block the DNA-binding activity of the CRISPR–Cas complex, yet do this by interacting with different protein subunits, and using steric or non-steric modes of inhibition. The third anti-CRISPR protein operates by binding to the Cas3 helicase–nuclease and preventing its recruitment to the DNA-bound CRISPR–Cas complex. In vivo, this anti-CRISPR can convert the CRISPR–Cas system into a transcriptional repressor, providing the first example—to our knowledge—of modulation of CRISPR–Cas activity by a protein interactor. The diverse sequences and mechanisms of action of these anti-CRISPR proteins imply an independent evolution, and foreshadow the existence of other means by which proteins may alter CRISPR–Cas function.

A New Group of Phage Anti-CRISPR Genes Inhibits the Type I-E CRISPR-Cas System of Pseudomonas aeruginosa

ABSTRACT CRISPR-Cas systems are one of the most widespread phage resistance mechanisms in prokaryotes. Our lab recently identified the first examples of phage-borne anti-CRISPR genes that encode protein inhibitors of the type I-F CRISPR-Cas system of Pseudomonas aeruginosa. A key question arising from this work was whether there are other types of anti-CRISPR genes. In the current work, we address this question by demonstrating that some of the same phages carrying type I-F anti-CRISPR genes also possess genes that mediate inhibition of the type I-E CRISPR-Cas system of P. aeruginosa. We have discovered four distinct families of these type I-E anti-CRISPR genes. These genes do not inhibit the type I-F CRISPR-Cas system of P. aeruginosa or the type I-E system of Escherichia coli. Type I-E and I-F anti-CRISPR genes are located at the same position in the genomes of a large group of related P. aeruginosa phages, yet they are found in a variety of combinations and arrangements. We have also identified functional anti-CRISPR genes within nonprophage Pseudomonas genomic regions that are likely mobile genetic elements. This work emphasizes the potential importance of anti-CRISPR genes in phage evolution and lateral gene transfer and supports the hypothesis that more undiscovered families of anti-CRISPR genes exist. Finally, we provide the first demonstration that the type I-E CRISPR-Cas system of P. aeruginosa is naturally active without genetic manipulation, which contrasts with E. coli and other previously characterized I-E systems. IMPORTANCE The CRISPR-Cas system is an adaptive immune system possessed by the majority of prokaryotic organisms to combat potentially harmful foreign genetic elements. This study reports the discovery of bacteriophage-encoded anti-CRISPR genes that mediate inhibition of a well-studied subtype of CRISPR-Cas system. The four families of anti-CRISPR genes described here, which comprise only the second group of anti-CRISPR genes to be identified, encode small proteins that bear no sequence similarity to previously studied phage or bacterial proteins. Anti-CRISPR genes represent a newly discovered and intriguing facet of the ongoing evolutionary competition between phages and their bacterial hosts. The CRISPR-Cas system is an adaptive immune system possessed by the majority of prokaryotic organisms to combat potentially harmful foreign genetic elements. This study reports the discovery of bacteriophage-encoded anti-CRISPR genes that mediate inhibition of a well-studied subtype of CRISPR-Cas system. The four families of anti-CRISPR genes described here, which comprise only the second group of anti-CRISPR genes to be identified, encode small proteins that bear no sequence similarity to previously studied phage or bacterial proteins. Anti-CRISPR genes represent a newly discovered and intriguing facet of the ongoing evolutionary competition between phages and their bacterial hosts.

Inactivation of CRISPR-Cas systems by anti-CRISPR proteins in diverse bacterial species.

CRISPR-Cas systems provide sequence-specific adaptive immunity against foreign nucleic acids1,2. They are present in approximately half of all sequenced prokaryotes3 and are expected to constitute a major barrier to horizontal gene transfer. We previously described nine distinct families of proteins encoded in Pseudomonas phage genomes that inhibit CRISPR-Cas function4,5. We have developed a bioinformatic approach that enabled us to discover additional anti-CRISPR proteins encoded in phages and other mobile genetic elements of diverse bacterial species. We show that five previously undiscovered families of anti-CRISPRs inhibit the type I-F CRISPR-Cas systems of both Pseudomonas aeruginosa and Pectobacterium atrosepticum, and a dual specificity anti-CRISPR inactivates both type I-F and I-E CRISPR-Cas systems. Mirroring the distribution of the CRISPR-Cas systems they inactivate, these anti-CRISPRs were found in species distributed broadly across the phylum Proteobacteria. Importantly, anti-CRISPRs originating from species with divergent type I-F CRISPR-Cas systems were able to inhibit the two systems we tested, highlighting their broad specificity. These results suggest that all type I-F CRISPR-Cas systems are vulnerable to inhibition by anti-CRISPRs. Given the widespread occurrence and promiscuous activity of the anti-CRISPRs described here, we propose that anti-CRISPRs play an influential role in facilitating the movement of DNA between prokaryotes by breaching the barrier imposed by CRISPR-Cas systems.

Protein Folding Kinetics Beyond the Φ Value: Using Multiple Amino Acid Substitutions to Investigate the Structure of the SH3 Domain Folding Transition State

The SH3 domain folding transition state structure contains two well-ordered turn regions, known as the diverging turn and the distal loop. In the Src SH3 domain transition state, these regions are stabilized by a hydrogen bond between Glu30 in the diverging turn and Ser47 in the distal loop. We have examined the effects on folding kinetics of amino acid substitutions at the homologous positions (Glu24 and Ser41) in the Fyn SH3 domain. In contrast to most other folding kinetics studies which have focused primarily on non-disruptive substitutions with Ala or Gly, here we have examined the effects of substitutions with diverse amino acid residues. Using this approach, we demonstrate that the transition state structure is generally tolerant to amino acid substitutions. We also uncover a unique role for Ser at position 41 in facilitating folding of the distal loop, which can only be replicated by Asp at the same position. Both these residues appear to accelerate folding through the formation of short-range side-chain to backbone hydrogen bonds. The folding of the diverging turn region is shown to be driven primarily by local interactions. The diverging turn and distal loop regions are found to interact in the transition state structure, but only in the context of particular mutant backgrounds. This work demonstrates that studying the effects of a variety of amino acid substitutions on protein folding kinetics can provide unique insights into folding mechanisms which cannot be obtained by standard Phi value analysis.

Long Noncontractile Tail Machines of Bacteriophages

In this chapter, we describe the structure, assembly, function, and evolution of the long, noncontractile tail of the siphophages, which comprise ∼60% of the phages on earth. We place -particular emphasis on features that are conserved among all siphophages, and trace evolutionary connections between these phages and myophages, which possess long contractile tails. The large number of high-resolution structures of tail proteins solved recently coupled to studies of tail-related complexes by electron microscopy have provided many new insights in this area. In addition, the availability of thousands of phage and prophage genome sequences has allowed the delineation of several large families of tail proteins that were previously unrecognized. We also summarize current knowledge pertaining to the mechanisms by which siphophage tails recognize the bacterial cell surface and mediate DNA injection through the cell envelope. We show that phages infecting Gram-positive and Gram-negative bacteria possess distinct families of proteins at their tail tips that are involved in this process. Finally, we speculate on the evolutionary advantages provided by long phage tails.

The Crystal Structure of Bacteriophage HK97 gp6: Defining a Large Family of Head-Tail Connector Proteins

The final step in the morphogenesis of long-tailed double-stranded DNA bacteriophages is the joining of the DNA-filled head to the tail. The connector is a specialized structure of the head that serves as the interface for tail attachment and the point of egress for DNA from the head during infection. Here, we report the determination of a 2.1 A crystal structure of gp6 of bacteriophage HK97. Through structural comparisons, functional studies, and bioinformatic analysis, gp6 has been determined to be a component of the connector of phage HK97 that is evolutionarily related to gp15, a well-characterized connector component of bacteriophage SPP1. Whereas the structure of gp15 was solved in a monomeric form, gp6 crystallized as an oligomeric ring with the dimensions expected for a connector protein. Although this ring is composed of 13 subunits, which does not match the symmetry of the connector within the phage, sequence conservation and modeling of this structure into the cryo-electron microscopy density of the SPP1 connector indicate that this oligomeric structure represents the arrangement of gp6 subunits within the mature phage particle. Through sequence searches and genomic position analysis, we determined that gp6 is a member of a large family of connector proteins that are present in long-tailed phages. We have also identified gp7 of HK97 as a homologue of gp16 of phage SPP1, which is the second component of the connector of this phage. These proteins are members of another large protein family involved in connector assembly.